Creating the “Coronavirus” 229E Genome

In order to generate an accurate genome of anything, that thing must be shown to exist in reality first. Regarding “viruses,” this means that the particles assumed to be “viruses” must be purified and isolated from everything else within the sample taken directly from a sick host. What is meant by purification and isolation?

PURIFICATION:

1. to make pure; free from anything that debases, pollutes, adulterates, or contaminates:
2. to free from foreign, extraneous, or objectionable elements:

https://www.dictionary.com/browse/purification

ISOLATION:

the act of sseparating something from other things : the act of isolating something

https://www.merriam-webster.com/dictionary/isolation

Only once the particles believed to be “viruses” are free of any contaminants/pollutants/foreign material/etc. and are separated from everything else, would it then be theoretically possible to get an accurate genome. These particles must come directly from a human and not be subjected to the cell culturing process as, in doing so, it automatically renders the final product a non-isolated and unpurified mess.

For more information on purification and isolation, please see the related posts here:

https://viroliegy.com/category/purification-isolation/

For added clarity on the invalid cell culture process, please refer to this post:

The Case Against Cell Cultures

Knowing this, it must be asked: are the genomes of supposed “viruses” coming from purified/isolated particles taken directly from sick patients? If “Coronavirus” 229E is any indication, the answer is a definite NO. Bear with me as outlining the creation of this genome is broken into a few parts and is a bit of a bumpy ride with a few detours along the way. Hopefully by the end some light will be shed on this subject.

The first part presents highlights from the 2012 “Coronavirus” 229E genome paper with a summary. Woven into the summary are two detours. The first one details the 2001 229E reference genome used to help build the 2012 version. The second detour offers highlights from a 2009 study said to outline the cell culture method utilized to create the 2012 229E genome. As the 2012 study left much of the important information out, these sections are meant to provide further insight into the insane steps and leaps in logic used by these researchers to acquire a genome from a “virus” never shown to physically exist in the first place.

The first complete genome sequences of clinical isolates of human coronavirus 229E

Abstract Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to insufficient pressure to maintain gene integrity of non-essential genes. We present here the first full-length genome sequence of two clinical isolates. Each encoded gene was compared to Inf-1. In general, little sequence changes were noted, most could be attributed to genetic drift, since the clinical isolates originate from 2009 to 2010 and VR740 from 1962. Hot spots of substitutions were situated in the S1 region of the Spike, the nucleocapsid gene, and the non-structural protein 3 gene, whereas several deletions were detected in the 30 UTR. Most notable was the difference in genome organization: instead of an ORF4A and ORF4B, an intact ORF4 was present in clinical isolates.”

“So far only one full genome has been determined for HCoV-229E [6]. This reference sequence is obtained from
the infectious HCoV-229E cDNA clone (Inf-1) that is based on the 1973-deposited laboratory-adapted prototype strain of HCoV-229E (VR-740). The 1973-deposited prototype strain was originally isolated in 1962 from a medical student with an upper respiratory infection at the University of Chicago [7, 8]. Of the current HCoV-229E isolates, only limited sequence data have been obtained. Chibu and Birch [5] have investigated the evolution of HCoV-229E by sequencing part of the S and the N gene from clinical samples collected between 1979 and 2004.

Sequence data from other genomic regions are still lacking, as no full-genome sequence of a non-lab-adapted virus is available.

Materials and methods

Clinical samples

We propagated several contemporary strains of HCoV-229E upon pseudostratified human airway epithelial. One of these, clinical strain HCoV-229E 0349 (21050349), was isolated from a respiratory swab collected in the Netherlands in 2010, from a stem cell transplantation recipient, who presented in hospital with fever and respiratory infection. The virus was propagated upon human airway epithelial cells, as described previously [9]. The apical
supernatant was harvested 72 h post-infection by apical washing. A second clinical isolate was uncultured, J0304, obtained from an adult with symptoms of lower respiratory tract infections in Italy, collected via the GRACE European Network of Excellence [10]. Ethics review committees in each country approved the study, and written informed consent was provided by all study participants.

Full-genome sequencing of the HCoV-229E clinical isolates

Total RNA was extracted from the apical washing from 0349 and from the Copan collected swab of J0304 (Copan Diagnostics) as described [11]. Reverse transcription was performed at 37 C for 1 h using random hexamers and superscript II (Invitrogen). The HCoV-229E Inf-1 reference sequence (Accession number NC_002645.1) was used as scaffold for designing bidirectional PCR–primer combinations, amplifying an average fragment length of 500 bp with a minimum overlap of 80 bp with adjacent primer combinations. Primers sequences are available upon request. Amplification of the fragments was performed with the following thermal cycle profile: 5 min at 95 C, 45 cycles of 95 C for 1 min, 55 C for 1 min, and 72 C for 2 min, followed by a final elongation step of 7 min at 72 C. PCR fragments were visualized upon agarose gel electrophoreses by ethidium bromide staining. Positive PCR fragments were directly sequenced with their forward and reverse primers in both the directions. Sequencing reactions were performed according to the BigDye Terminator v1.1 protocol (ABI life science). Sequences were analyzed with Coloncode Aligner software (version 3.7.1). Sequences have been submitted to GenBank (JX503060, JX503061).

5′ and 3′ RACE

In order to complete the full-genome sequence with 5′ and 3′ termini, 5′ and 3′ RACE was performed. The 5′ end was
determined with the 5′ RACE kit (Invitrogen) according to the manufactures protocol. Gene-specific primers for 5′ RACE PCR amplification were designed to flank approximately 100 nt of the 5′ region. The 3′ end of HCoV-229E clinical strain was determined with 3′ RACE, with an RT
reaction performed using the Oligo-dT-JZH primer and PCR amplification with the JZH primer and a gene-specific primer [9].The PCR products were excised after agarose
electrophoresis and purified with the Nucleospin Extract II kit (Machery-Nagel) according to the manufacture protocol. Purified PCR products were cloned into the pCRII-TOPO TA vector (invitrogen) and chemically competent E. Coli according to manufacture protocol (Top 10 cells, Invitrogen). Transformants were directly analyzed via colony PCR with T7 and M13Rev primers. PCR products were sequenced as described above.

Full-genome sequence analysis

The ZCURVE_CoV1.0 program was used to recognize and predict putative proteins coding genes [12, 13]. Phylogenic analyses (neighbor-joining method) were conducted using MEGA, version 4.02. The identity between HCoV-229E clinical isolates and the reference sequence of 229E (inf-1NC_002645.1) was investigated by pairwise alignment using BioEdit Sequence Aligner. Simplot (version 3.5.1) was used to draw similarity/distance plots. N- and O-linked glycosylation sites and signal peptide cleavage sites were predicated using the NetNGly 1.0, NetOGly 3.1, and SignalP 4.0 analysis tools, from the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/). The identity comparisons per gene were investigated by pairwise alignment using BioEdit Sequence Aligner.”

“The putative S proteins of 0349 and J0304 contain, respectively, 27 and 26 potential N-glycosylation sites upon analysis with the NetNgly 1.0 analysis tool, from the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/), while the reference sequence contains 24 potential N-glycosylation sites. Indeed these 24 are conserved and there are three extra at positions 20, 111, and 488 in isolate 0349, and in J0304 there are two extra at positions 111 and 488. The predicted signal peptide of S in the reference sequence is present at amino acids 1–16, using the SignalP 4.0 tool from the Center for Biological Sequence Analysis. The predicted signal peptide of both clinical isolates is also located at amino acids 1–16, with potential cleavage site between 16 and 17.”

“A phylogenetic analysis which includes our clinical isolates, the reference sequence, and various S sequences from clinical samples collected between 1979 and 2004 provides further evidence of divergence in time as shown previously (Fig. 1a) [5]. Chibo and Birch presented four phylogenetically distinct S gene sequences of which the clustering matched with the year of isolation, indicating genetic drift in time. Isolate 0349 and J0304 cluster with the group 4 viruses, a group that contains all GenBank S-sequences that have been collected from 1999 onwards.

The ORF4 gene is located between the spike and envelope gene. Its function is unknown, yet studies with HCoV-NL63 accessory protein ORF3, a homolog of 229E-ORF4, revealed that the protein is incorporated into virions and is, therefore, an additional structural protein [13]. There are major differences between the clinical isolates and the reference sequence. Most important is a 2 nt deletion in the reference strain resulting in an interruption of the gene, whereas the two clinical isolates have an intact ORF4, as previously published [20]. The putative full protein is 219 amino acids in size. Besides the insertion/deletion 17 nt changes are observed (equal for both clinical isolates), resulting in nine amino acid substitutions.”

Evaluation of the 30 UTR reveals significant variation. In the clinical isolate 0349, there are 8 nt substitutions and three deletions observed. In J0304, there are nine substitutions and two deletions. The largest deletion in both isolates is a 38 nt deletion. Furthermore, two short deletions with lengths of 2 and 4 nt are observed in 0349, and a 4 nt deletion in J0304. The effect of such deletions on 30 UTR structure and function is unknown. For the betacoronaviruses MHV and BCoV, it has been proposed that there are two conserved RNA structures at the upstream end of the
30 UTR: a bulged stem-loop and an adjacent pseudoknot [22].”

In this study, we report the first full-genome sequences of two non-laboratory adapted strains. Alignment of nucleotide and protein sequences and phylogenetic analysis of the two HCoV-229E strains showed several differences with the reference sequence. Genetic drift was noticed in the spike gene, and the only part of the genome truly affected by lab-adaptation is the ORF4 gene.”

doi: 10.1007/s11262-012-0807-9

In Summary:

• Human “coronavirus” 229E was identified in the mid-1960s, yet only one full-genome sequence was available
• This full-length sequence had been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740
• Lab-adaptation might have resulted in genomic changes, due to insufficient pressure to maintain gene integrity of non-essential genes
• The researchers claimed that little sequence changes were noted, most could be attributed to the genetic drift theory, since the clinical isolates originate from 2009 to 2010 and VR740 from 1962
• So far only one full genome has been determined for HCoV-229E as described in reference 6

DETOUR #1:

In order to understand what was done to create the 2012 229E genome, two detours highlighting reference studies need to be made to shed some further light on this process.

The first detour details the REFERENCE GENOME used in order to create the new genome. First, a quick definition of a reference genome:

Reference Genome

“A reference genome (also known as a reference assembly) is a digital nucleic acid sequence database, assembled by scientists as a representative example of the set of genes in one idealized individual organism of a species. As they are assembled from the sequencing of DNA from a number of individual donors, reference genomes do not accurately represent the set of genes of any single individual organism. Instead a reference provides a haploid mosaic of different DNA sequences from each donor. There are reference genomes for multiple species of viruses, bacteria, fungus, plants, and animals.”

https://en.m.wikipedia.org/wiki/Reference_genome

A reference genome is considered the most idealized representation of an entity and is used as a template to build future genomes. This template is utilized to map the DNA of the organism being sequenced. In other words, a genome is only as good and as accurate as the reference genome used to build it from in the first place.

In the case of “Coronavirus” 229E, the only reference genome available came from a cDNA-clone Inf-1 of 229E from 2001 based upon a lab-adapted strain from 1973 which is itself based on an “isolate” from 1962. The process for it’s creation is detailed in reference 6:

However, before we jump into reference 6, to better understand this part, it is best to freshen up on the definition of recombinant first:

Recombinant Definition

“of or resulting from new combinations of genetic material:

the genetic material produced when segments of DNA from different sources are joined to produce recombinant DNA.”

https://www.dictionary.com/browse/recombinant

“a: relating to or containing genetically engineered DNA

b: produced by genetic engineering”

https://www.merriam-webster.com/dictionary/recombinant

In other words, recombinant “viruses” are lab-created, genetically engineered “viruses” containing DNA from various sources.

Now that we have all of that out of the way, on to highlights from reference 6:

INFECTIOUS RNA TRANSCRIBED IN VITRO FROM A cDNA COPY OF THE HUMAN CORONAVIRUS GENOME CLONED IN VACCINIA VIRUS

“Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes.”

“Despite the remarkable achievements of Almazán et al. (2000)R1 and Yount et al. (2000)R35 , we have been unable to construct a stable, full-length cDNA copy of the genome of either the human coronavirus strain 229E (HCoV) or murine hepatitis virus (MHV) using plasmids, bacterial artificial chromosomes, bacteriophage vectors or an in vitro approach based upon long-range RT–PCR (Thiel et al., 1997R32 ; Herold et al., 1998R7 ). We, therefore, decided to pursue an alternative strategy based upon the optimization of in vitro DNA ligation, the use of vaccinia virus as a eukaryotic cloning vector and the cytoplasmic expression of transfected RNA that has been transcribed in vitro. We reasoned that this approach would have several advantages. Firstly, poxvirus vectors are eminently suitable for the cloning of large cDNAs. It has been shown that they have the capacity to accept at least 26 kbp of foreign DNA (Smith & Moss, 1983R29 ) and recombinant vaccinia genomes of this size are stable, infectious and replicate in tissue culture to the same titre as non-recombinant virus. Secondly, vaccinia virus vectors have been developed that are designed for the insertion of foreign DNA by in vitro ligation (Merchlinsky & Moss, 1992R19 ). This obviates the need for plasmid intermediates carrying the entire cDNA insert. Thirdly, using this approach, recombinant virus is recovered from an infectious RNA that is introduced and replicates in the cytoplasm of the transfected cell. Thus, there are no concerns regarding RNA modification, processing and export from or degradation within the nucleus.

In this study, we show that human coronavirus cDNA fragments of more than 27 kbp can be stably cloned and propagated in vaccinia virus. Moreover, a recombinant vaccinia virus clone, containing a full-length HCoV cDNA, enabled us to produce infectious in vitro RNA transcripts and to rescue recombinant human coronavirus.

Methods

▪ Cells and virus and RNA transfection. Human lung fibroblast (MRC-5), monkey kidney fibroblast (CV-1) and human cervix epithelial (HeLa-S3) cells were purchased from the European Collection of Cell Cultures and maintained in minimum essential medium (MEM) supplemented with HEPES (25 mM), foetal bovine serum (5–10%) and antibiotics. HCoV 229E, vaccinia virus strain vNotI/tk (Merchlinsky & Moss, 1992R19 ) and vaccinia virus recombinants were propagated, titrated and purified by using standard procedures (Raabe et al., 1990R24 ; Mackett et al., 1985R13 ). Fowlpox virus strain HP1.441 (Mayr & Malicki, 1966R17 ) was propagated in chicken embryo fibroblast cells that were maintained in MEM supplemented with 7% foetal bovine serum.”

Discussion

“The system we describe here should find wide application in the analysis of the molecular biology and pathogenesis of coronaviruses. We have shown that it is possible to clone a full-length cDNA copy of the human coronavirus genome in the vaccinia virus genome and to produce infectious RNA transcripts from this template. In the long term, this system will improve our ability to control coronavirus infections in humans, livestock and domestic animals.”

“Secondly, the system we describe will complement existing methods of producing recombinant coronaviruses (Masters, 1999R15 ; Almazán et al., 2000R1 ; Yount et al., 2000R35 ) and significantly advance the analysis of coronavirus pathogenesis. With the systems now available, it should be possible to generate rapidly a large collection of genetically modified coronaviruses; for example, intra- and interspecific chimeric viruses, viruses with gene inactivations or deletions and viruses with attenuating modifications or supplementary functions. The phenotypes associated with these modifications, at least those that are not lethal, can then be tested in animal models of infection. In particular, this should provide important insights into the relationship between coronavirus infection and the immune response.”

https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-82-6-1273#tab2

In order to create the 229E genome in 2001:

• A reverse-genetic system was created for the generation of recombinant “coronaviruses”
• This system was based upon the in vitro transcription of infectious RNA from a cDNA copy of the human “coronavirus” 229E genome that has been cloned and propagated in vaccinia “virus”
• They decided to pursue an alternative strategy based upon the optimization of in vitro DNA ligation, the use of vaccinia “virus” as a eukaryotic cloning vector and the cytoplasmic expression of transfected RNA that has been transcribed in vitro
• They did so for three reasons:
  1. “Poxvirus” vectors are eminently suitable for the cloning of large cDNAs as it had been shown that they have the capacity to accept at least 26 kbp of foreign DNA and recombinant vaccinia genomes of this size are stable, infectious and replicate in tissue culture to the same titre as “non-recombinant virus”
  2. Vaccinia “virus” vectors were developed that were designed for the insertion of foreign DNA by in vitro ligation
  3. Using this approach, recombinant “virus” was said to be recovered from an infectious RNA that was introduced and replicated in the cytoplasm of the transfected cell
• In this study, the researchers claim to show that human “coronavirus” cDNA fragments of more than 27 kbp can be stably cloned and propagated in vaccinia “virus”
• Moreover, a recombinant vaccinia “virus” clone, containing a full-length HCoV cDNA, enabled them to produce infectious in vitro RNA transcripts and to rescue recombinant human “coronavirus”
• Human lung fibroblast (MRC-5), monkey kidney fibroblast (CV-1) and human cervix epithelial (HeLa-S3) cells were purchased from the European Collection of Cell Cultures and maintained in minimum essential medium (MEM) supplemented with HEPES (25 mM), foetal bovine serum (5–10%) and antibiotics
• HCoV 229E, vaccinia “virus” strain vNotI/tk and vaccinia “virus” recombinants were propagated, titrated and purified by using standard procedures
• Fowlpox “virus” strain HP1.441 was propagated in chicken embryo fibroblast cells that were maintained in MEM supplemented with 7% foetal bovine serum
• Feel free to read the rest of the paper which details the many steps involved in creating this “coronavirus” genome from various different sources (vaccinia “virus,” recombinant vaccinia “virus,” fowlpox “virus”) outside of a “coronavirus” but the bottom line is that they cloned a full-length cDNA copy of the human “coronavirus” genome in the vaccinia “virus” genome to produce infectious RNA transcripts from this template

Thus, the “idealized” reference genome used for the creation of the 229E genome in 2012 is nothing but a synthetic lab-created cloned recombinant mixture of DNA from many sources. It did not come directly from purified/isolated “virus” particles taken from a human.

END DETOUR # 1.

• This reference sequence is obtained from the infectious HCoV-229E cDNA clone (Inf-1) that is based on the 1973-deposited laboratory-adapted prototype strain of HCoV-229E (VR-740)
• The 1973-deposited prototype strain was originally “isolated” in 1962 from a medical student with an upper respiratory infection at the University of Chicago
• Of the current HCoV-229E isolates, only limited sequence data have been obtained
• Sequence data from other genomic regions are still lacking, as no full-genome sequence of a non-lab-adapted “virus” was available
• The “virus” was propagated upon human airway epithelial cells, as described in reference 9

DETOUR # 2:

To better understand the cell culture process they used for the 2012 genome, the researchers referenced this 2009 study on the HBoV as the method they used:

Human Bocavirus Can Be Cultured in Differentiated Human Airway Epithelial Cells

“In 2005, a human bocavirus was discovered in children with respiratory tract illnesses. Attempts to culture this virus on conventional cell lines has failed thus far. We investigated whether the virus can replicate on pseudostratified human airway epithelium. This cell culture system mimics the human airway environment and facilitates culturing of various respiratory agents. The cells were inoculated with human bocavirus-positive nasopharyngeal washes from children, and virus replication was monitored by measuring apical release of the virus via real-time PCR. Furthermore, we identified different viral mRNAs in the infected cells. All mRNAs were transcribed from a single promoter but varied due to alternative splicing and alternative polyadenylation, similar to what has been described for bovine parvovirus and minute virus of canines, the other two members of the Bocavirus genus. Thus, transcription of human bocavirus displays strong homology to the transcription of the other bocaviruses. In conclusion, we report here for the first time that human bocavirus can be propagated in an in vitro culture system and present a detailed map of the set of mRNAs that are produced by the virus.”

“Presently, the Bocavirus genus includes bovine parvovirus (BPV), minute virus of canines (MVC), and the recently identified human bocavirus (HBoV) and HBoV type 2 (2, 9, 15). HBoV was identified in 2005 within pools of human nasopharyngeal aspirates obtained from individuals with respiratorytract illnesses. The current genomic DNA reference sequence of HBoV is 5,299 nt in length, but sequence information regarding the flanking terminal hairpin structures remains to be determined (2). HBoV has been found worldwide, mainly in respiratory samples, but in some cases, HBoV has also been detected in serum, fecal samples, and urine samples (1, 16, 20, 31). HBoV infections are frequently diagnosed in 2-year-old children with upper or lower respiratory tract illness, often in combination with another respiratory virus (2, 16, 20). One of the most frequently observed clinical symptoms in HBoV-infected patients is acute wheezing (1). Despite the current knowledge regarding HBoV, no in vitro or in vivo model that supports replication of HBoV has been established.

In this study, we investigated whether pseudostratified human airway epithelium cell culture could be utilized as a model for HBoV replication. Pseudostratified epithelium is formed by culturing primary human airway epithelial cells in an air-liquid interface. The morphology and functionality of the cells resemble those of the human airways, and this system has been used previously to culture a wide range of respiratory viruses, e.g., influenza virus (28), parainfluenza virus (33), respiratory syncytial virus (34), adenovirus (21), and severe acute respiratory syndrome coronavirus (26). In this study, we documented HBoV replication upon inoculation with respiratory material
from HBoV-infected patients. We observed apical release of virus and analyzed the viral mRNA transcripts in the infected cells.

Human airway epithelial cell culture. Cryopreserved human trachea epithelial
primary cells (HTEpC) were obtained from the European Collection of Cell Cultures. HTEpC were maintained for one serial passage as a monolayer in bronchial/tracheal epithelial cell serum-free growth medium (European Collection of Cell Cultures) supplemented with penicillin-streptomycin. Bronchial/
tracheal epithelial cell serum-free growth medium was refreshed every 2 or 3 days. HTEpC cultures were maintained at 37°C in a 5% CO2 incubator. When reaching 75% confluence, cells were dissociated with 2 ml of TrypLE Express enzyme (Invitrogen). HTEpC were diluted in air-liquid interface medium (11), which is a mixture of LHC basal medium (Invitrogen) and Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with the required additives (Sigma). A total of 8 104 HTEpC were seeded on type IV collagen (Sigma)-coated 12-well ThinCerts with a 0.4-m pore size (Greiner Bio-One). Medium was renewed every 2 or 3 days. When cultures reached full confluence, the cells were exposed to air. HTEpC cultures on the air-liquid interface were maintained in 12-well deep-well plates (Greiner Bio-One) for 21 days to let the cells differentiate into pseudostratified human airway epithelial cell cultures. Medium from the basolateral compartment was renewed every 6 days, and the apical surface was washed every 2 days with Hanks’ balanced salt solution (HBSS) (Invitrogen).

HBoV infection. An aliquot of 50 l clinical patient material was diluted in 200 l HBSS and centrifuged for 30 min at 4°C with 10,000 relative centrifugal force. Two hundred microliters of diluted clinical sample was directly inoculated upon the apical surface of pseudostratified human airway epithelium and incubated for 2 h at 34°C in a 5% CO2 incubator. After 2 h, 200-l samples were collected from both the apical and basolateral sides. Inoculated cultures were maintained at 34°C in a 5% CO2 incubator. Samples were collected after 24, 48, 72, and 95 h postinoculation (hpi) from both the apical and basolateral sides. Apical washing and harvesting was performed by adding 200 l of HBSS to the apical surface and incubation for 10 min at 34°C in a 5% CO2 incubator, followed by the removal and storage of the 200 l HBSS from the apical surface. Washing is needed to remove the mucus, which will otherwise suffocate the cells. An aliquot of 50 l apical wash was transferred into 900 l L6 lysis buffer (6) for HBoV DNA quantification. At the last day of culture, the cells were collected in TRIzol reagent (Invitrogen) for HBoV mRNA analysis. Cultures were transferred to a conventional 12-well plate (Greiner Bio-One) and analyzed by eye with a phase-contrast microscope, prior to cell collection in TRIzol reagent.”

RESULTS

HBoV infection of human airway epithelial cultures. Currently, there is no culture system that supports HBoV replication. Pseudostratified human airway epithelium, which is formed after exposure of human tracheal epithelial cells to air, is the best imitation of the human trachea. We tested whether this system allows propagation of HBoV. Clinical material from three HBoV-infected patients was inoculated at the apical side of pseudostratified human airway epithelium and incubated for 2 h at 34°C. The culture was maintained, and 200-l harvests were collected from the apical and basolateral sides at 2, 24, 48, 72, and 95 hpi.”

“To investigate the cytopathic effect of HBoV infection, we monitored the morphological changes of the cells at 95 hpi with the Bonn-1 isolate by phase-contrast microscopy. Minor changes in the culture were noticed. Stretching of cells (which was absent in the control culture) was observed, but there was no disruption of the pseudostratified epithelium layer and the ciliated cell density. Furthermore, the ciliary movement and the mucosal secretion of the Bonn-1 virus-inoculated culture were similar to those of the control culture.”

DISCUSSION

This is the first study that presents a culture system for HBoV. Pseudostratified human airway epithelium cell culture can be used as a model for HBoV replication. We observed virus replication after inoculation with a nasopharyngeal washing from an HBoV-infected patient. Apical secretion of HBoV was observed at 72 hpi, but there were no obvious morphological changes to the cells as seen for respiratory syncytial virus (34).

The difficulty of isolating respiratory viruses from clinical material on conventional cell lines has been described in the mid-1960s, leading to ex vivo culturing of human embryo respiratory tract explants (29, 30). Nowadays, the usage of human embryo respiratory tract explants raises ethical issues, but efforts to isolate HBoV from clinical respiratory material on conventional cell lines, like LLC-MK2, HEp-2, Vero, and MRC-5 cells were not successful (10, 19, 35). It is likely that these cell lines are not susceptible to certain respiratory viruses, e.g., because they are deficient in receptor expression or no longer exhibit their cell-specific phenotypes, such as basal,
secretory, and ciliated cells.”

doi: 10.1128/JVI.00614-09.

• In 2005, a human “bocavirus” was discovered in children with respiratory tract illnesses
• Attempts to culture this “virus” on conventional cell lines had failed (which raises the question of how this “virus” was discovered)
• The researchers investigated whether the “virus” can replicate on pseudostratified human airway epithelium
• This cell culture system is said to mimic the human airway environment and facilitates culturing of various respiratory agents
• HBoV was identified in 2005 within pools of human nasopharyngeal aspirates obtained from individuals with respiratorytract illnesses
• The current genomic DNA reference sequence of HBoV is 5,299 nt in length, but sequence information regarding the flanking terminal hairpin structures remains to be determined
• In other words, HBoV is nothing but an unfinished sequence created from nasal secretions of multiple children which were mixed together
• Despite the current knowledge regarding HBoV, no in vitro or in vivo model that supports replication of HBoV had been established
• In this study, they investigated whether pseudostratified human airway epithelium cell culture could be utilized as a model for HBoV replication.
• Pseudostratified epithelium is formed by culturing primary human airway epithelial cells in an air-liquid interface

CULTURE METHOD USED:

This is an outline of the process they used for HBoV which was later referenced as what was used to create the 2012 229E genome. As the 2012 study did not detail the exact steps, we can only conclude it was similar to what is described here:

1. Cryopreserved human trachea epithelial primary cells (HTEpC) were obtained from the European Collection of Cell Cultures and were maintained for one serial passage as a monolayer in bronchial/tracheal epithelial cell serum-free growth medium supplemented with penicillin-streptomycin
2. Bronchial/tracheal epithelial cell serum-free growth medium was refreshed every 2 or 3 days
3. HTEpC cultures were maintained at 37°C in a 5% CO2 incubator
4. When reaching 75% confluence, cells were dissociated with 2 ml of TrypLE Express enzyme (Invitrogen)
5. HTEpC were diluted in air-liquid interface medium (11), which is a mixture of LHC basal medium (Invitrogen) and Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with the required additives (Sigma)
6. A total of 8  104 HTEpC were seeded on type IV collagen (Sigma)-coated 12-well ThinCerts with a 0.4-m pore size (Greiner Bio-One)
7. Medium was renewed every 2 or 3 days
8. When cultures reached full confluence, the cells were exposed to air
9. HTEpC cultures on the air-liquid interface were maintained in 12-well deep-well plates (Greiner Bio-One) for 21 days to let the cells differentiate into pseudostratified human airway epithelial cell cultures
10. Medium from the basolateral compartment was renewed every 6 days, and the apical surface was washed every 2 days with Hanks’ balanced salt solution (HBSS) (Invitrogen)

• There was no culture system that supports HBoV replication
• Pseudostratified human airway epithelium, which is formed after exposure of human tracheal epithelial cells to air, is the best imitation of the human trachea
• To investigate the cytopathic effect of HBoV infection, they monitored the morphological changes of the cells at 95 hpi with the Bonn-1 isolate by phase-contrast microscopy
• Minor changes in the culture were noticed
• Stretching of cells (which was absent in the control culture) was observed, but there was no disruption of the pseudostratified epithelium layer and the ciliated cell density
• Furthermore, the ciliary movement and the mucosal secretion of the Bonn-1 “virus-inoculated” culture were similar to those of the control culture
• In other words, they noticed barely any CPE changes between the “virus” and control cultures
• They determined that pseudostratified human airway epithelium cell culture can be used as a model for HBoV replication
• Apical secretion of HBoV was observed at 72 hpi, but there were no obvious morphological changes to the cells as seen for respiratory syncytial “virus”
• The difficulty of isolating respiratory “viruses” from clinical material on conventional cell lines has been described in the mid-1960s
• Efforts to isolate HBoV from clinical respiratory material on conventional cell lines, like LLC-MK2, HEp-2, Vero, and MRC-5 cells were not successful
• It is likely that these cell lines are not susceptible to certain respiratory “viruses,” e.g., because they are deficient in receptor expression or no longer exhibit their cell-specific phenotypes, such as basal, secretory, and ciliated cells

In this 2009 study, the researchers admit that they could not propagate/replicate HBoV in any cell line and had to create a system mimicking the human trachea in order to produce it. However, upon looking for the cytopathogenic effect (CPE), an indicator used to indirectly determine “virus,” there was minimal to no CPE observed. Oddly enough, they still claimed the method a success mostly based on genomic data/analysis.

If one were to look at this study honestly, it is apparent that the researchers did not propagate a “virus” at all utilizing this method. Still, it is the method that was chosen in order to culture the material for the new 2012 version of the 229E genome. While this 2009 study took us a little bit out of the way, it needed to be outlined in order to show the ridiculous cell culture steps required to create the 2012 genome. It is clear that these researchers are incapable of purifying/isolating “virus” material directly from sick humans so they must synthetically create it in a lab.

END DETOUR # 2.

• The 2012 researchers propagated several contemporary strains of HCoV-229E upon pseudostratified human airway epithelial cells, as described previously in reference [9]
• The apical supernatant was harvested 72 h post-infection by apical washing
• Two strains were used:
  • The first clinical strain HCoV-229E 0349 (21050349), was “isolated” from a respiratory swab collected in the Netherlands in 2010, from a stem cell transplantation recipient, who presented in hospital with fever and respiratory infection
  • The second clinical isolate was uncultured, J0304, obtained from an adult with symptoms of lower respiratory tract infections in Italy
• Total RNA was extracted from the apical washing from 0349 and from the Copan collected swab of J0304 as described in reference 11 (which described a protocol for the small-scale “purification” of DNA and RNA from human serum and urine, not NP swabs or human airway epithelial cell cultures)
• The 2001 HCoV-229E Inf-1 reference sequence was used as scaffold for designing bidirectional PCR–primer combinations, amplifying an average fragment length of 500 bp with a minimum overlap of 80 bp with adjacent primer combinations
• Amplification of the fragments was performed with the following thermal cycle profile: 5 min at 95 C, 45 cycles of 95 C for 1 min, 55 C for 1 min, and 72 C for 2 min, followed by a final elongation step of 7 min at 72 C
• “Purified” PCR products were cloned into the pCRII-TOPO TA vector (invitrogen) and chemically competent E. Coli according to manufacture protocol
• The ZCURVE_CoV1.0 program was used to recognize and predict putative (i.e commonly accepted or supposed; assumed to exist or to have existed) proteins coding genes
• The identity between 2012 HCoV-229E clinical isolates and the 2001 reference sequence of 229E was investigated by pairwise alignment using BioEdit Sequence Aligner
• Simplot (version 3.5.1) was used to draw similarity/distance plots
• N- and O-linked glycosylation sites and signal peptide cleavage sites were predicated using the NetNGly 1.0, NetOGly 3.1, and SignalP 4.0 analysis tools, from the Center for Biological Sequence Analysis
• The identity comparisons per gene were investigated by pairwise alignment using BioEdit Sequence Aligner
• The putative S proteins of 0349 and J0304 contain, respectively, 27 and 26 potential N-glycosylation sites upon analysis with the NetNgly 1.0 analysis tool while the 2001 reference sequence contains 24 potential N-glycosylation sites
• The predicted signal peptide of S in the 2001 reference sequence was present at amino acids 1–16, using the SignalP 4.0 tool while the predicted signal peptide of both 2012 clinical isolates was also located at amino acids 1–16, with potential cleavage site between 16 and 17
• A phylogenetic analysis which includes their clinical isolates, the 2001 reference sequence, and various S sequences from clinical samples collected between 1979 and 2004 provided further evidence of divergence in time (i.e. they couldn’t sequence the same genome twice)
• The researchers claimed there were major differences between the clinical isolates and the 2001 reference sequence
• Most important is a 2 nt deletion in the 2001 reference strain resulting in an interruption of the gene, whereas the two 2012 clinical isolates have an intact ORF4
• Evaluation of the 30 UTR revealed significant variation
• In the clinical isolate 0349, there are 8 nt substitutions and three deletions observed while in J0304, there are nine substitutions and two deletions
• The largest deletion in both isolates is a 38 nt deletion
• Furthermore, two short deletions with lengths of 2 and 4 nt were observed in 0349, and a 4 nt deletion in J0304
• The effect of such deletions on 30 UTR structure and function is unknown
• The researchers report the first full-genome sequences of two non-laboratory adapted strains
• Alignment of nucleotide and protein sequences and phylogenetic analysis of the two HCoV-229E strains showed several differences with the 2001 reference sequence

When breaking down the creation of the 2012 “coronavirus” 229E genome, it is clear that there was no attempt at purification/isolation of any “virus” before it was assembled. The researchers continued to utilize the same cell culture tricks of mixing human DNA with foreign animal DNA, antibiotics, fetal bovine serum, chemicals, “nutrients,” additives, etc. They looked for a genome from within the unpurified cell culture soup and utilized the reference genome from a 1973 cloned recombinant lab-adapted creation as a map to create their new one. The genomes differed in many areas yet instead of realizing that these were nothing but artificial creations stemming from wildly different procedures, the researchers decided that these differences were due to antigenic drift (i.e. random genetic mutation over time).

None of this comes as a surprise when you realize that the original 229E “virus” was never properly purified/isolated directly from a sick patient nor proven pathogenic by fulfilling Koch’s Postulates when Dorothy Hamre first “discovered” it in the 1960’s. The “coronavirus” has never been proven to exist in reality. It only “exists” as random A,C,T,G’s in a computer database taken from a cell culture soup stitched together on the back of a recombinant cloned synthetic creation.

For more on the original “discovery” of 229E, please see this post:

https://viroliegy.com/2021/12/06/hamres-coronavirus-229e-paper-1966/